Project Summary Transmembrane water channels known as aquaporins (AQPs) play significant roles in maintaining transparency, biomechanics, refractive index gradient (RING) and homeostasis in the avascular mammalian lens. To get nourishment and eliminate metabolic wastes, the lens creates a microcirculatory current involving AQPs, ion- and solute transporters, and cotransporters. Mutations in fiber cell-specific AQP0, and knockout (KO) of the gene resulted in lens cataract whereas those of epithelial cell-specific AQP1 did not cause any obvious defects in mouse lens under normal physiological conditions. However, lenses of AQP1 KO and AQP5 KO mice developed osmotic swelling and cataract under hyperglycemic stress conditions. Several lens proteins such as beaded filament proteins (CP49 and filensin), crystallins and connexins interact with AQP0, possibly to modulate its functions. The goal here is to elucidate the mechanisms by which the three AQPs, AQP0, AQP1 and AQP5, play important roles to elicit and maintain lens transparency, RING and homeostasis under normal and stressful conditions (such as diabetes). Our long term objective is to contribute to the prevention and treatment of cataracts. The main hypothesis is: Aquaporins play critical roles in the lens microcirculation, biomechanics, RING and osmoregulation, and alterations in their function(s) lead to cataracts. The Specific Aims are to: 1. To explore whether a combination of a decreasing gradient of intact and an increasing gradient of end cleaved forms of AQP0, from the cortex to the nucleus, is required for maintaining lens transparency, biomechanics and RING in vivo. 2. To test whether both water permeability and CTCA functions are critical for maintaining lens transparency, biomechanics and RING, as well as, to explore in vitro, in vivo and ex vivo the molecular mechanism by which AQP0 exerts CTCA between the lens fiber cells. 3. To investigate if regulation of AQP0, AQP1 and AQP5 plays significant roles in maintaining lens osmoregulation and RING for transparency, biomechanics and homeostasis under normal and diabetic hyperglycemic stress conditions. The objectives will be pursued using structure-function approach, as appropriate, and performing microscopy, cell culture, organ culture as well as cytological, biomechanical, biochemical, physiological, and molecular biological experiments along with developing an animal model. The proposed studies have the potential to gather significant data and key information on the mechanistics of the roles played by AQPs in lens transparency and homeostasis. The results could offer new directions for novel therapeutic strategies for the treatment of cataracts and other aquaporin-related diseases in the eye, such as dry eye, glaucoma, retinal detachment, macular degeneration and retinopathy.